The use of fluorides has proven to have a positive effect on the
prevention of tooth decay and has been considered one of the most
important public health measures of the 20th century. (1-2) Its
administration (which can be topical or systemic) aims to maintain a
constant concentration of the fluoride ion (F) in the oral cavity to
facilitate the incorporation of these crystals on the surface of the
erupted enamel--decreasing demineralization rate and increasing
remineralization rate--. (3) However, currently it is known that the
excessive ingestion of F- has deleterious effects on enamel development,
generating a hypomineralized porous phenotype with reduced hardness. (4)
In addition to the aesthetic and functional consequences, an in-vitro
study conducted at Unidad de Investigacion en Caries (UNICA) in
extracted teeth with moderate fluorosis from Colombian patients,
suggests that the porosity of the enamel with fluorosis makes it more
susceptible to demineralization. (5)

Unlike the etiologic factor of dental fluorosis--which is fully
identified as the chronic exposure to high concentrations of F- between
the ages of 0 and 5 years--, (6) little is known about the cellular and
molecular mechanisms affected by F- and leading to the development of
fluorosis. In Colombia, there are epidemiological reports (7) that
identify the sources of intake of F- possibly responsible for the high
prevalence, (8,9) but the pathogenesis of the defect has not been
properly investigated.

We know that the chronic and sustained presence of the F- ion in
plasma increases the likelihood of adhering to tissues in the
mineralization process, (10) but there is the misperception that the
hypomineralization observed in dental fluorosis is the only consequence
of the excessive addition of F- in enamel. In this literature review, we
assume that the hypomineralized phenotype of fluorosis is the outcome of
a series of possible effects of the F-ion on cell physiology and the
proteins responsible for guiding the mineralization of enamel
(biomineralization). First, we will make a brief introduction to the
normal process of enamel biomineralization, and then we will study the
available evidence on the effects of F- during key moments of the
process.

Amelogenesis and enamel biomineralization

Enamel is a nano-compound bioceramics (11) with 95% inorganic
material, 4% water, and 1% organic matter. (12, 13) Four elements are
required for the formation of enamel: cell, ions, proteins, and a
compartment where the mineralization reaction takes place (extracellular
matrix). (12) The entire process of enamel formation is called
amelogenesis, while the mineralization process as such, which takes
place between ions and proteins secreted by cells, is called
biomineralization. The enamelforming cells are called ameloblasts, and
during the process they go through a series of changes that are
summarized in the following stages: differentiation, secretion,
maturation, and transition. (14) During the phase of secretion of
ameloblasts, they carry ions from the plasma into the interior of the
cell: the type of ion that enters will depend, among other factors, on
its availability in the plasma at the moment. (15) This is why the
mineral of enamel (which is similar to the pure mineral hydroxyapatite)
usually contains a variety of ions, such as HP[O.sup.2-.sub.4],
C[O.sup.2-.sub.3], [Na.sup.+] y F-. (16)

In addition to transporting and secreting ions, ameloblasts
synthesize and secrete a large amount of proteins, which are the major
component of enamel in formation (> 90%). (17) Among the secreted
proteins are first those of the extracellular matrix (amelogenin,
ameloblastin, and enamelin) and secondly the proteases:
metalloproteinase matrix 20 (MMP-20) and kallikrein 4 (KLK-4). The
enamelin and the ameloblast in function as nucleators, attracting ions
to their protein structure to favor the organized deposit of crystals of
calcium phosphate salts. (18)

On the other hand, the amelogenin takes a higher supramolecular
organization (20 nm nanospheres) and works as a "scaffold"
that guides the growth of crystals for the formation of prisms. (19) The
role of ameloblastin is less clear, but it has been found to have
functions in the adhesion and control of ameloblasts differentiation.
(20) The MMP-20 gradually and selectively degrades the protein support
during the secretion and maturation stages, to allow the widening of the
enamel crystals which previously grew in length. (21) At the start of
the maturation stage, cells stop producing MMP-20 and begin to produce
KLK-4, a protease that completes the process of degradation of enamel
protein material. (22) The KLK-4 cuts the remains of structural proteins
into small peptides that can be processed in the cell. (23) At the end
of this orderly process of the maturation stage, the protein component
will be less than 1% (11) and the resulting enamel will have minimum
porosity and a translucent shiny look with a smooth feel.

Based on the evidence about the normal mechanisms of enamel
formation (summarized above), several studies on the induction of
fluorosis have been conducted to understand which steps of the process
are affected by fluoride, as described below.

What concentrations of fluoride are used in in vivo and in vitro
experiments for the induction of dental fluorosis?

Despite the existence of standardized models (described below),
there is no consensus as to the concentration of F- biologically
relevant to induce dental fluorosis in in vivo and in vitro studies. On
the one hand, a line of evidence in the studies uses micromolar
([micro]M) concentrations of F- and considers that concentrations of
2-12 [micro]mol/L are biologically relevant. (24-27) Another line of
evidence argues that, under normal conditions, there are basal levels of
F- in the fluid of enamel which proportionally increase in the presence
of concentrations of F- in plasma and expose the ameloblasts to
milimolar concentrations (mM) of F-. (28) The difficulties for a
consensus and the directly proportional relationship between the dose of
F- and cellular responses make it necessary to examine the effects
reported for Falong with the concentration used in the experiments.

In vivo and in vitro models standardized for the study of dental
fluorosis

Two in vitro models have been established: the first one consists
of a line of immortalized cells similar to ameloblasts, resulting from
the enamel organ of the first molar in newborn Swiss-Webster mice. This
line was achieved by transfection of cells in the epithelium of the
enamel organ with the oncogene SV40 virus and has been named LS8. (29)
The second model consists of standardized primary cultures of epithelial
cells of the enamel organ of human fetuses of 21 weeks of age. (30) The
latter model represents the ideal in vitro model for the study of
fluorosis, as it comes from human cells and expresses more bookmarks
than the mouse line; however, sampling has ethical implications and
technical difficulties that make the LS8 cell model the more convenient
and the most widely used.

As to in vivo models, some mammals have been used, including rats,
(31) mice, (32) hamsters, (33) rabbits (34) and higher species, such as
pigs (35) and sheep. (36) The rat model has proven to be the most
appropriate for the study of dental fluorosis, (31) since the incisors
of rodents erupt continuously, and a single tooth can show the different
stages of enamel development; in addition, there is evidence that the
levels of F-plasma required for the appearance of fluorotic defects in
enamel are very similar in humans and other animals. (25, 31,37, 38)

At the molecular level, dental fluorosis is a consequence of the
delay in the removal of proteins from the extracellular matrix, mainly
during enamel maturation.

The induction of fluorosis in animal and cell models has enabled to
determine the extent to which the F-circulating in plasma in excessive
concentrations during amelogenesis has deleterious effects on the
different stages, including the secretion stage. (39) It has been
reported that at this stage fluoride induces alterations in the
vesicular transport of ameloblasts (40) and in the intracellular
degradation of proteins of the matrix by the lysosomal system. (41, 42)
However, experimental studies on fluorosis have focused specifically on
the stage of enamel maturation (which includes the orderly sequence of
crystal growth, proteolytic digestion by different enzymes, and
absorption of protein residues), as it has proven to be the most
sensitive to the negative effects of F-. This is based on in vivo
studies performed in rats, which have demonstrated that the consumption
of high amounts of F- delays the elimination of proteins (especially
amelogenins). (24,25) A reduced capacity of amelogenin elimination
triggered by F- prevents the thickening of enamel crystals and leads to
incomplete mineralization. (43)

In the same stage of maturation, in which the ameloblasts regulate
pH by secreting bicarbonate and using ionic transporters to absorb
protons from the matrix, (44) the large number of protons ([H.sup.+])
released as a result of the high rate of precipitation of enamel
crystals produce fluctuations of pH (from neutral to acid), (45) and the
presence of high concentrations of F- in an acidic environment has
deleterious effects, as will be discussed later in this review. While
the effect of F- on maturation is critical, its adverse effect on the
other stages is not negligible and could be cumulative, considering that
the severity of the fluorosis is linked to a sustained and prolonged
exposure. (43)

Effects of fluoride on the physiology of ameloblasts

It has been reported that a concentration of 10 [micro]M of F-
produces a decrease in the expression of MMP20, (46), 47 concentrations
of 10 to 20 [micro]M of F- produce an increase in apoptosis, and
concentrations above 1 mM produce alterations in cell proliferation.
(47) In addition, concentrations of 120 [micro]M of F- reduce the
expression of messengers of amelogenin, ameloblastin, enamelin, and
MMP-20, as well as factors of vascularization, such as the endothelial
growth factor (VEGF), monocyte chemoattractant proteins (MCP-1) and the
interferon inducible protein (IP-10). (48)

The pH regulation is fundamental for crystal growth: the
precipitation of ions during enamel maturation releases a large amount
of protons ([H.sup.+]), followed by reaction [10[Ca.sup.2+] + 6
HP[O.sup.2-.sub.4] + 2[H.sub.2]O [Ca.sub.10]
[(P[O.sub.4]).sub.6][(OH).sub.2] + 8[H.sup.+]], and the pH of the
extracellular matrix goes from neutral to slightly acidic. (45) These pH
changes are reflected in the alteration of the morphology of these
cells, which show rough ends in the presence of an acid pH and smooth
ends in the presence of a neutral pH. During the normal development of
enamel there is an alternation of these morphologies; however, in vitro
studies have shown that the modulation between smooth and rough
morphology become slow, with predominance of the rough one. (47)
therefore, as a result of the presence of fluoride, the pH of the
extracellular matrix remains acid for a long time.

The exposure of LS8 cells to concentrations of 250-2000 [micro]M of
F- has provided interesting observations: the increase in the
concentration of protons ([H.sup.+]), in the presence of a high
concentration of ions F-, leads to the formation of hydrofluoric acid
(HF), which is absorbed by the cells and causes severe changes in
cellular metabolism. (49) These findings suggest an interesting
hypothesis: an excess in cytoplasmic F- in ameloblast induces stress in
the endoplasmic reticulum and activates a defense called "unfolded
protein response" (UPR), which decreases the synthesis and
secretion of KLK-4, essential for the elimination of the protein matrix
of amelogenin and the final maturation of enamel prisms. (50-52)

When F- passes through the cytoplasmic membrane toward the
mineralization front, another chain of effects, demonstrated by recent
in vitro studies describing the massive arrival of F- in the
mineralization front, produces a hypermineralized layer of enamel which
could act as a physical barrier that would prevent the diffusion of ions
and proteins to the subsurface of the mineralization front. (53) This
molecular event could hinder the entry of "raw material"
necessary for full mineralization of crystals and thus contribute to the
hypominealization of fluorotic enamel.

Effects of fluoride on the activity of proteases of the
extracellular matrix

It is widely known that F- inhibits the activity of proteases of
the extracellular matrix of enamel. Since the number of studies is
limited, the results have failed to demonstrate a direct inhibition of
the enzyme activity, (54-56) and therefore F- has been discarded as an
inhibitor of proteases in the pathogenesis of fluorosis.

Effects of fluoride on the kinetics of biomineralization

The incorporation of F- in enamel crystals during their formation
increases the binding strength of amelogenin to the crystal (57) and
reduces its hydrolysis. (58) This evidence has been gathered in trials
with recombinant amelogenin bound to synthetic hydroxyapatite with F-
concentrations like those found in human teeth with fluorosis. The
binding of proteins to crystals with high content of F- can possibly
trigger changes in their conformation, thus "hiding" some
cleavage sites and decreasing access to the proteases, (57) reducing the
speed of removal of matrix proteins and preventing the thickening and
maturation of the crystal. These studies provide sufficient evidence to
suggest that dental fluorosis is a consequence of the delay in the
removal of proteins during the stage of maturation of enamel. In
addition, proteins may possibly be retained in erupted enamel.

Based on this logic, some studies have been conducted on the
retention of amelogenin in erupted enamel with fluorosis. (58, 59) The
low protein content of erupted enamel (< 1% in healthy enamel) and
the difficulties to extract proteins trapped in the mineral matrix have
limited the studies aimed at extracting, identifying, and quantifying
the protein material of enamel.

In order to expand the study of dental fluorosis in Colombia, at
the Unidad de Investigacion en Caries (UNICA) we standardized a method
to extract and identify enamel proteins through liquid chromatography
along with mass spectrometry. The method was applied to a sample of
teeth from Colombian patients. (60) Using this method, we compared the
proteins identified in erupted enamel of healthy teeth and teeth with
fluorosis. Our analysis showed amelogenin, ameloblastin, and
enamelin--the latter more frequently in fluorotic enamel, suggesting a
possible role of this protein in the events that trigger fluorosis--. In
addition, through the relative quantification of identified peptides of
amelogenin, we found no differences in protein content between healthy
enamel and enamel with fluorosis. (11) The available reports on this
subject are contradictory, (58, 59, 61) and to date we cannot speak of
retention of proteins in fluorotic enamel but of an alteration in speed
for their removal, which slows down the maturation process of enamel
crystal. (43)

Figure 1 summarizes the available evidence on the cellular and
biochemical mechanisms reported to date, possibly related to the
pathogenesis of dental fluorosis.

CONCLUSIONS AND EXPECTATIONS

The cellular and molecular mechanisms by which dental fluorosis
occurs have not been fully explained. Nor is there consensus on the
biologically relevant concentrations of F- that produce dental fluorosis
in humans. In vitro and in vivo models have shown that high steady
concentrations of F- have harmful effects on ameloblasts. These
deleterious effects are proportional to the doses of F- used and
decreases the capacity of ameloblasts to synthesize and secrete
proteins, especially at the maturation stage. The susceptibility of this
stage in particular may be due to pH fluctuations experienced by
ameloblasts due to a high concentration of protons released during the
precipitation of crystals. While F- has been thought to be a direct
inhibitor of MMP-20 and KLK-4 proteases (as a possible cause of protein
retention), the available evidence to date allows discarding this
hypothesis. For now, it is widely known that F- affects the kinetics of
biomineralization, slows down the hydrolysis of proteins, and interrupts
the process of elimination of the protein matrix, triggering the
incomplete mineralization of enamel crystals and producing porous
enamel--which is typical of dental fluorosis.

Further studies on the pathogenesis of dental fluorosis are
expected to provide evidence to the analysis of biologically relevant
concentrations of fluoride (e.g., fluoride in plasma of inhabitants from
fluorosis-endemic areas) and thus align them to in vivo and in vitro
studies. It is also important, under such concentrations, to carry out
studies on other effects of F- on the cellular physiology and the
kinetics of biomineralization in vitro to fully elucidate the mechanisms
that lead to this defect and to replicate studies that--in the presence
of contradictory evidence--confirm whether the enamel with fluorosis
shows retention of proteins.

ACKNOWLEDGEMENTS

To Dr. Margarita Usuga Vacca for her critical review of the
manuscript.